S-Nitroso- N-acetyl-L-cysteine ethyl ester (SNACET) and N-acetyl-L-cysteine ethyl ester (NACET)-Cysteine-based drug candidates with unique pharmacological profiles for oral use as NO, H2S and GSH suppliers and as antioxidants: Results and overview

Abstract

S-Nitrosothiols or thionitrites with the general formula RSNO are formally composed of the nitrosyl cation (NO⁺) and a thiolate (RS^-), the base of the corresponding acids RSH. The smallest S-nitrosothiol is HSNO and derives from hydrogen sulfide (HSH, H₂S). The most common physiological S-nitrosothiols are derived from the amino acid L-cysteine (CysSH). Thus, the simplest S-nitrosothiol is S-nitroso-L-cysteine (CysSNO). CysSNO is a spontaneous potent donor of nitric oxide (NO) which activates soluble guanylyl cyclase to form cyclic guanosine monophosphate (cGMP). This activation is associated with multiple biological actions that include relaxation of smooth muscle cells and inhibition of platelet aggregation. Like NO, CysSNO is a short-lived species and occurs physiologically at concentrations around 1 nM in human blood. CysSNO can be formed from CysSH and higher oxides of NO including nitrous acid (HONO) and its anhydride (N₂O₃). The most characteristic feature of RSNO is the S-transnitrosation reaction by which the NO⁺ group is reversibly transferred to another thiolate. By this way numerous RSNO can be formed such as the low-molecular-mass S-nitroso-N-acetyl-L-cysteine (SNAC) and S-nitroso-glutathione (GSNO), and the high-molecular-mass S-nitrosol-L-cysteine hemoglobin (HbCysSNO) present in erythrocytes and S-nitrosol-L-cysteine albumin (AlbCysSNO) present in plasma at concentrations of the order of 200 nM. All above mentioned RSNO exert NO-related biological activity, but they must be administered intravenously. This important drawback can be overcome by lipophilic charge-free RSNO. Thus, we prepared the ethyl ester of SNAC, the S-nitroso-N-acetyl-L-cysteine ethyl ester (SNACET), from synthetic N-acetyl-L-cysteine ethyl ester (NACET). Both NACET and SNACET have improved pharmacological features over N-acetyl-L-cysteine (NAC) and S-nitroso-N-acetyl-L-cysteine (SNAC), respectively, including higher oral bioavailability. SNACET exerts NO-related activities which can be utilized in the urogenital tract and in the cardiovascular system. NACET, with high oral bioavailability, is a strong antioxidant and abundant precursor of GSH, unlike its free acid N-acetyl-L-cysteine (NAC). Here, we review the chemical and pharmacological properties of SNACET and NACET as well as their analytical chemistry. We also report new results from the ingestion of S-[¹⁵N]nitroso-N-acetyl-L-cysteine ethyl ester (S¹⁵NACET) demonstrating the favorable pharmacological profile of SNACET.

Keywords: CGMP; Nitric oxide; Oxidative stress; Pharmaceuticals; S-Nitrosothiols.

Fig. 1

Chemical structures of NACET and its major metabolites in biological systems. Formal substitution of the H (proton) of the SH group by NO (nitrosyl) yields the corresponding S-nitrosothiols.

Fig. 2

Time-dependent inhibition of the activity (prostaglandin E₂ formation from 10 µM arachidonic acid) of recombinant COX-1 (5 U) by NACET and acetylsalicylic acid (ASA) at the indicated concentrations. Data are indicated as mean±SEM from three analyses. The experimental conditions and the measurement of prostaglandin E₂ were described previously . Under the same conditions and concentrations, SNACET was found to increase COX-1 activity up to 125%, while salicylic acid did not alter COX-1 activity at all (data not shown).

Fig. 3

(A) Inhibition of collagen-induced aggregation of washed human platelets by SNACET and SNCET at the indicated concentrations. Data are indicated as mean±SD from three independent measurements. (B) Inhibition of collagen-induced aggregation of washed human platelets and thromboxane A₂ (TxA₂) synthesis by SNACET and SNCET at the indicated concentrations. Data are indicated as mean±SD from four independent measurements. The collagen concentration was 2 µg/µL in both experiments. Human platelets were isolated as described elsewhere. Platelet aggregation and the measurement of thromboxane B₂ (TxB₂), the stable reaction product of TxA₂, were performed as described previously , .

Fig. 4

(A) Pharmacokinetics of [¹⁵N]nitrate (Na¹⁵NO₃, 99 atom% ¹⁵N; Sigma, Germany) at the dose of 101 µmol [¹⁵N]nitrate/kg bodyweight, or [¹⁵N]nitrite (Na¹⁵NO₂, 99 atom% ¹⁵N; Aldrich, Germany) at the dose of 48.4 mmol [¹⁵N]nitrite/kg bodyweight in two male rabbits. (B) Pharmacokinetics of S-[¹⁵N]nitroso-N-acetylcysteine ethyl ester (S¹⁵NACET) in four male rabbits at the doses of 63, 68, 72 and 126 µmol S¹⁵NACET/kg bodyweight. The weight of the rabbits ranged between 1.8–4.8 kg. All substances were administered together with D(+)-glucose (220 mg) in 2 mL of drinking water via a stomach probe. Administered S¹⁵NACET was free of [¹⁵N]nitrite and [¹⁵N]nitrate. Rabbits were treated once only. The first blood sample was taken immediately before drug administration. Horizontal lines at the value of 1.0 indicate the theoretical baseline molar ratio in rabbit plasma. The study was approved by the local supervisory committee for studies in animal (Hannover, Germany). The y axes indicate the molar ratio of [¹⁵N]nitrate/[¹⁵N]nitrite in the rabbit plasma relative to the unlabeled nitrate (i.e., [¹⁴N]nitrate) and unlabeled nitrite ([¹⁴N]nitrite).

Fig. 5

Pharmacokinetics and pharmacodynamics of synthetic S¹⁵NACET and sodium [¹⁵N]nitrate in a male healthy subject. (A) Effects of orally administered S¹⁵NACET (1 µmol/kg bodyweight), sodium [¹⁵N]nitrate (1 µmol/kg bodyweight) or drinking water free of synthetic S¹⁵NACET and [¹⁵N]nitrate on creatinine-corrected urinary excretion rate of cGMP (in nmol/mmol). (B) Effect of orally administered S¹⁵NACET (1 µmol/kg bodyweight) on creatinine-corrected urinary excretion rate of [¹⁵N]nitrate and [¹⁵N]nitrite. S¹⁵NACET and [¹⁵N]nitrate were taken orally as their dilutions in 200 mL drinking water (indicated by the arrows) immediately after preparation of their stock solutions and collection of the first urine sample. Administered S¹⁵NACET was free of [¹⁵N]nitrite and [¹⁵N]nitrate. Note the different units used for [¹⁵N]nitrate (in µmol/mmol creatinine) and [¹⁵N]nitrite (in nmol/mmol creatinine). The [¹⁵N]nitrate-to-[¹⁵N]nitrite molar ratio ranged between 44:1 and 1167:1, indicating [¹⁵N]nitrate as the major S¹⁵NACET metabolite across the entire observation time window.